Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Jun;9(6):1210–1216. doi: 10.1110/ps.9.6.1210

Overall rotational diffusion and internal mobility in domain II of protein G from Streptococcus determined from 15N relaxation data.

M L Tillett 1, M J Blackledge 1, J P Derrick 1, L Y Lian 1, T J Norwood 1
PMCID: PMC2144651  PMID: 10892813

Abstract

The backbone dynamics and overall tumbling of protein G have been investigated using 15N relaxation. Comparison of measured R2/R1 relaxation rate ratios with known three-dimensional coordinates of the protein show that the rotational diffusion tensor is significantly asymmetric, exhibiting a prolate axial symmetry. Extensive Monte Carlo simulations have been used to estimate the uncertainty due to experimental error in the relaxation rates to be D(parallel)/D(perpendicular) = 1.68 +/- 0.08, while the dispersion in the NMR ensemble leads to a variation of D(parallel)/D(perpendicular) = 1.65 +/- 0.03. Incorporation of this tensorial description into a Lipari-Szabo type analysis of internal motion has allowed us to accurately describe the local dynamics of the molecule. This analysis differs from an earlier study where the overall rotational diffusion was described by a spherical top. In this previous analysis, exchange parameters were fitted to many of the residues in the alpha helix. This was interpreted as reflecting a small motion of the alpha helix with respect to the beta sheet. We propose that the differential relaxation properties of this helix compared to the beta sheet are due to the near-orthogonality of the NH vectors in the two structural motifs with respect to the unique axis of the diffusion tensor. Our analysis shows that when anisotropic rotational diffusion is taken into account NH vectors in these structural motifs appear to be equally rigid. This study underlines the importance of a correct description of the rotational diffusion tensor if internal motion is to be accurately investigated.

Full Text

The Full Text of this article is available as a PDF (264.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barchi J. J., Jr, Grasberger B., Gronenborn A. M., Clore G. M. Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy. Protein Sci. 1994 Jan;3(1):15–21. doi: 10.1002/pro.5560030103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cordier F., Caffrey M., Brutscher B., Cusanovich M. A., Marion D., Blackledge M. Solution structure, rotational diffusion anisotropy and local backbone dynamics of Rhodobacter capsulatus cytochrome c2. J Mol Biol. 1998 Aug 14;281(2):341–361. doi: 10.1006/jmbi.1998.1950. [DOI] [PubMed] [Google Scholar]
  3. Farrow N. A., Muhandiram R., Singer A. U., Pascal S. M., Kay C. M., Gish G., Shoelson S. E., Pawson T., Forman-Kay J. D., Kay L. E. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 1994 May 17;33(19):5984–6003. doi: 10.1021/bi00185a040. [DOI] [PubMed] [Google Scholar]
  4. Kay L. E. Protein dynamics from NMR. Nat Struct Biol. 1998 Jul;5 (Suppl):513–517. doi: 10.1038/755. [DOI] [PubMed] [Google Scholar]
  5. Kay L. E., Torchia D. A., Bax A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry. 1989 Nov 14;28(23):8972–8979. doi: 10.1021/bi00449a003. [DOI] [PubMed] [Google Scholar]
  6. Kelley L. A., Gardner S. P., Sutcliffe M. J. An automated approach for defining core atoms and domains in an ensemble of NMR-derived protein structures. Protein Eng. 1997 Jun;10(6):737–741. doi: 10.1093/protein/10.6.737. [DOI] [PubMed] [Google Scholar]
  7. Kördel J., Skelton N. J., Akke M., Palmer A. G., 3rd, Chazin W. J. Backbone dynamics of calcium-loaded calbindin D9k studied by two-dimensional proton-detected 15N NMR spectroscopy. Biochemistry. 1992 May 26;31(20):4856–4866. doi: 10.1021/bi00135a017. [DOI] [PubMed] [Google Scholar]
  8. Lian L. Y., Derrick J. P., Sutcliffe M. J., Yang J. C., Roberts G. C. Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance. J Mol Biol. 1992 Dec 20;228(4):1219–1234. doi: 10.1016/0022-2836(92)90328-h. [DOI] [PubMed] [Google Scholar]
  9. Lian L. Y., Yang J. C., Derrick J. P., Sutcliffe M. J., Roberts G. C., Murphy J. P., Goward C. R., Atkinson T. Sequential 1H NMR assignments and secondary structure of an IgG-binding domain from protein G. Biochemistry. 1991 Jun 4;30(22):5335–5340. doi: 10.1021/bi00236a002. [DOI] [PubMed] [Google Scholar]
  10. Luginbühl P., Pervushin K. V., Iwai H., Wüthrich K. Anisotropic molecular rotational diffusion in 15N spin relaxation studies of protein mobility. Biochemistry. 1997 Jun 17;36(24):7305–7312. doi: 10.1021/bi963161h. [DOI] [PubMed] [Google Scholar]
  11. Mandel A. M., Akke M., Palmer A. G., 3rd Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J Mol Biol. 1995 Feb 10;246(1):144–163. doi: 10.1006/jmbi.1994.0073. [DOI] [PubMed] [Google Scholar]
  12. Palmer A. G., 3rd Probing molecular motion by NMR. Curr Opin Struct Biol. 1997 Oct;7(5):732–737. doi: 10.1016/s0959-440x(97)80085-1. [DOI] [PubMed] [Google Scholar]
  13. Schurr J. M., Babcock H. P., Fujimoto B. S. A test of the model-free formulas. Effects of anisotropic rotational diffusion and dimerization. J Magn Reson B. 1994 Nov;105(3):211–224. doi: 10.1006/jmrb.1994.1127. [DOI] [PubMed] [Google Scholar]
  14. Tillett M. L., Horsfield M. A., Lian L. Y., Norwood T. J. Protein-ligand interactions measured by 15N-filtered diffusion experiments. J Biomol NMR. 1999 Mar;13(3):223–232. doi: 10.1023/a:1008301324954. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES